We thank Elsevier for their authorization (license number 4334770630127) to reproduce here Figure 2A from Unterreiner et al. (2017).

Experiments were conducted according to the guidelines and standards of the Novartis Human Research Ethics Committee.
1. Preparation of Peripheral Blood Mononuclear Cells (PBMCs) from Human Buffy Coats
NOTE: We received buffy coats from healthy volunteers one day after collection, in 50 mL bags. They were provided under informed consent and collected through the Interregionale Blutspende Schweizeriches Rotes Kreuz. We handled them using the procedure below, at room temperature unless specified otherwise.
<ol>
	<li>Prepare a sterile and clean pair of scissors and a 1 L beaker with a plastic bag (under a lamina flow).</li>
	<li>Transfer the buffy coat into the beaker and carefully open it with the scissors.</li>
	<li>Using a 25 mL pipette, add 100 mL of phosphate-buffered saline buffer/ethylenediaminetetraacetic acid (PBS/EDTA: PBS 1x pH 7.4 containing no CaCl2 and no MgCl2, supplemented with 2 mM EDTA pH 8.0). 
	NOTE: Using the same pipette and after slowly pipetting the solution up and down, dispense 25 mL of the diluted buffy coat into 6 conical centrifuge tubes of 50 mL pre-filled with 15 mL of polysaccharide-based density gradient.</li>
	<li>Centrifuge 20 min at 800 x g with moderate acceleration (set at 4 out of 9) and without brake to allow for separation of cells based on their densities. 
	NOTE: After centrifugation, three layers will be visible; a pellet containing red blood cells and granulocytes, an upper layer made of plasma, and in between a white ring containing peripheral blood mononuclear cells (PBMCs).</li>
	<li>Harvest the PBMC ring using a 10 mL pipette and transfer to new 50 mL tubes. 
	NOTE: Two to three 50 mL tubes are normally required per buffy coat. At this stage, some plasma likely contaminates the collected PBMCs, which should have no impact on the subsequent enrichment steps.</li>
	<li>Top up to 50 mL every tube using PBS/EDTA and proceed to three successive washes with decreasing centrifugation time and speed (15 min at 520 x g, 10 min at 330 x g, 8 min at 150 x g). 
	NOTE: At each step, the supernatant is poured off in a liquid waste container and the cell pellet is resuspended in 50 mL of PBS/EDTA (pellets may be pooled after the first wash).</li>
	<li>After the final wash, resuspend the pellet in 25 mL of an ice-cold lysis buffer (see Table of Materials) to lyse the red blood cells by osmotic pressure.</li>
	<li>Incubate until the solution becomes clear (&#8804;5 min at room temperature).</li>
	<li>Stop the reaction by adding 25 mL of separation buffer (PBS 1x pH 7.4 containing no CaCl2 and no MgCl2, supplemented with 2% heat-inactivated fetal bovine serum (FBS) and 1 mM EDTA pH 8.0).</li>
	<li>Wash one more time at 150 x g for 8 min. 
	NOTE: At this stage PBMCs can be used as a bulk population (see step 3: PBMCs and monocytes treatments and stimulatory conditions) or processed for monocyte enrichment (see step 2: Preparation of monocytes from PBMCs), or frozen down for subsequent use (see step 5: Monocytes and PBMCs freezing/thawing procedures).</li>
</ol>
2. Preparation of Monocytes from PBMCs
<ol>
	<li>Resuspend the PBMCs obtained at step 1.10 in separation buffer and count them to reach 5 x 107cells/mL.</li>
	<li>Transfer the cell suspension into a 14 mL conical centrifuge tube with cap.</li>
	<li>Add 50 &#181;L of monocyte enrichment antibody cocktail per mL of cell suspension, vortex, and incubate 10 min at 4 &#176;C.</li>
	<li>Add 50 &#181;L of monocyte enrichment beads per mL of cells. 
	NOTE: The beads must be thoroughly vortexed to ensure homogeneity of the suspension.</li>
	<li>After adding the beads, shortly vortex the cell suspension and incubate 5 min at 4 &#176;C.</li>
	<li>Rinse the upper part of the tube with separation buffer until the tube is filled up to 10 mL.</li>
	<li>Slowly mix the solution by pipetting up and down.</li>
	<li>Place the tube without cap into a separation magnet.</li>
	<li>Incubate for 2.5 min at room temperature.</li>
	<li>Pour slowly into a 15 mL conical centrifuge tube.</li>
	<li>At this stage, use monocytes directly (see step 3: PBMCs and monocytes treatments and stimulatory conditions) or differentiate them into immature Monocyte-derived Dendritic Cells (iMoDCs) (see step 4), or freeze them down for subsequent use (see step 5).</li>
</ol>
3. PBMCs and Monocytes Treatments and Stimulatory Conditions
<ol>
	<li>Count cells and dilute them in culture medium (Roswell Park Memorial Institute medium (RPMI) 10% FBS + 1 mM Sodium Pyruvate + 100 U/mL Penicillin Streptomycin (Pen/Strep) + 5 &#181;M &#946;-mercaptoethanol), down to 1.25 x 104 cells/well.</li>
	<li>Distribute 30 &#181;L of cell suspension per well of a 384-well plate.</li>
	<li>Add 15 &#181;L of 4x concentrated compound solutions and pre-incubate for 1 h at 37 &#176;C, 5% CO2.</li>
	<li>Add lipopolysaccharide (LPS) to a final concentration of 10 ng/mL, or depleted zymosan (DZ) to a final concentration of 100 &#181;g/mL, or keep in plain medium.</li>
	<li>Incubate overnight at 37 &#176;C, 5% CO2.</li>
	<li>Take 10 &#181;L of the supernatant to measure secreted TNF-a levels (see step 7: Cytokines and viability measurements).</li>
</ol>
4. Monocytes Differentiation into iMoDCs and Stimulatory Conditions
<ol>
	<li>Count monocytes (enriched at step 2) and centrifuge the cell suspension at 520 x g for 5 min.</li>
	<li>Pipette off the supernatant and add culture medium (RPMI+ 10% FBS to obtain a final cell suspension of 0.4 x 106 cells/mL.</li>
	<li>Add 80 ng/mL recombinant human IL-4 + 100 ng/mL GM-CSF.</li>
	<li>Dispense 5 mL of cell suspension per well in a 6-well plate.</li>
	<li>Incubate for 7 days at 37 &#176;C, 5% CO2.</li>
	<li>At day 7, harvest cells by pipetting gently to avoid their activation.</li>
	<li>Centrifuge at 520 x g for 5 min.</li>
	<li>Vacuum aspirate and resuspend in 50 mL of culture medium without growth factors.</li>
	<li>Centrifuge at 520 x g for 5 min and resuspend at 1 x 106 cells/mL in culture medium.</li>
	<li>Dispense 100 &#181;L of cell suspension (105 cells) per well of a 96-well flat bottom plate.</li>
	<li>Pre-incubate for 1 h at 37 &#176;C after adding 50 &#181;L of 4x concentrated compound solutions, prepared as described at step 8: Compounds preparation.</li>
	<li>Add 50 &#181;L of stimuli (4x concentrated), prepared as described at step 9.</li>
	<li>Incubate 24 h at 37 &#176;C.</li>
	<li>Mix each well and transfer cells and supernatant (SN) into a 96-well V-bottom plate.</li>
	<li>Spin down at 475 x g for 5 min.</li>
	<li>Transfer supernatant into a new 96-well flat bottom plate, seal and freeze at -20 &#176;C until further use.</li>
</ol>
5. Monocytes and PBMCs Freezing/Thawing Procedures
<ol>
	<li>Freezing
	<ol>
		<li>Spin down PBMC or monocyte cell preparations at 520 x g for 5 min.</li>
		<li>Vacuum aspirate the supernatant and resuspend cells in freezing medium at 1 x 107 cells/mL.</li>
		<li>Dispense 1 mL of cell suspension into cryotubes, transfer the tubes to a specific cooling device (see Materials table) and place it at -80&#160;&#176;C.</li>
	</ol>
	</li>
	<li>Thawing
	<ol>
		<li>Thaw the cryotube and quickly transfer its content into a 15 mL conical centrifuge tube containing 9 mL of culture medium.</li>
		<li>Centrifuge at 520 x g for 5 min.</li>
		<li>Vacuum aspirate supernatant and resuspend cell pellet in 5 mL of culture medium. Cells are now ready for further experimental processing.</li>
	</ol>
	</li>
</ol>
6. Mouse Spleen Cells Preparation and Treatment
NOTE: We conducted animal sacrifices according to the guidelines and standards of the Novartis Animal Welfare Organization. Studies were approved by the Ethics Committee of the regional governmental authority (Kantonales Veterin&#228;ramt der Stadt Basel). We sacrificed animals by isoflurane over-exposure, with all efforts made to minimize suffering.
<ol>
	<li>Harvest the spleen and dissociate tissue using a tube equipped with a mechanical tissue grinder device and filled with 5 mL of cold RPMI medium.</li>
	<li>Use the spleen program of the dissociation machine to grind organs.</li>
	<li>Filter the cells through a 100 mm nylon cell strainer.</li>
	<li>Transfer the suspension into 50 mL tubes and centrifuge at 4 &#176;C for 10 min at 320 x g.</li>
	<li>Vacuum aspirate the supernatant, resuspend the cell pellet in 3 mL of ice-cold lysis buffer, and incubate for &#8804;2 min on ice.</li>
	<li>Stop lysis by adding 7 mL of RPMI medium.</li>
	<li>Filter again through a 100 &#181;m nylon cell strainer.</li>
	<li>Spin down cell suspension at 330 x g for 10 min at 4 &#176;C.</li>
	<li>Vacuum aspirate supernatant and resuspend cells at 11 x 106 cells/mL in complete medium (RPMI supplemented with 10% FBS, 100 U/mL Pen/Step and 5 &#181;M &#946;-Mercaptoethanol).</li>
	<li>Plate 1 x 106 cells/well (90 &#181;L) in a 96-well plate (flat bottom).</li>
	<li>Add 5 &#181;L of 20x concentrated compound solution previously diluted in RPMI medium as described in step 8.3: Serial dilution for murine spleen cells.</li>
	<li>Incubate for 30 min at 37 &#176;C, 5% CO2.</li>
	<li>Add 5 &#181;L of 20x concentrated DZ (final concentration 30 &#181;g/mL) or 20x concentrated LPS + IFN-g (TLR-4) (final concentration 1 &#181;M LPS and 10 ng/mL IFN-g).</li>
	<li>Incubate overnight at 37 &#176;C, 5% CO2.</li>
	<li>Centrifuge at 330 x g for 10 min.</li>
	<li>Transfer supernatants in new plates, seal and freeze at -20 &#176;C until further use.</li>
</ol>
7. Cytokines and Viability Measurements
<ol>
	<li>Human TNF-a measurement by HTRF (Homogenous Time Resolved Fluorescence) 
	NOTE: The protocol followed the supplier&#8217;s recommendations, briefly summarized below.
	<ol>
		<li>Mix 1 volume of reconstituted reagent (anti TNF-a-cryptate and anti-TNF-a-XL665) with 19 volumes of reconstitution buffer (50 mM phosphate buffer pH 7.0, 0.8 M potassium fluoride (KF), 0.2% bovine serum albumin (BSA)).</li>
		<li>Mix the two ready-to-use antibody solutions 1:1 just prior to dispensing the reagents.</li>
		<li>Dispense 10 &#181;L of supernatants from step 3.6 into white 384-well plates.</li>
		<li>Dispense 10 &#181;L of the antibody mix.</li>
		<li>Cover the plate with a sealer and incubate at 4 &#176;C overnight.</li>
		<li>Read plate on a microplate reader (50&#8211;200 flash).</li>
	</ol>
	</li>
	<li>Human IL-23 measurement by HTRF (Homogenous Time Resolved Fluorescence) 
	NOTE:&#160;The protocol followed the supplier&#8217;s recommendations, briefly summarized below.
	<ol>
		<li>Mix one volume of reconstituted reagent (anti IL-23-cryptate-antibody and anti-IL-23 D2-antibody) with 19 volumes of detection buffer #3.</li>
		<li>Mix the two ready-to-use antibody solutions 1:1 just prior to dispensing the reagents.</li>
		<li>Dispense 10 &#181;L of supernatants from step 3.6 into white 384-well plates.</li>
		<li>Dispense 10 &#181;L of the antibody mix.</li>
		<li>Cover the plate with a sealer and incubate at 4 &#176;C overnight.</li>
		<li>Read plate on a microplate reader (50&#8211;200 nm flash).</li>
	</ol>
	</li>
	<li>Human IL-6, IL-8, IL-1&#946; and TNF-&#945; measurements by electrochemiluminescence 
	NOTE: All samples were diluted at 1/150 in diluent 2 (first dilution: 10 &#181;L in 150 &#181;L, then 20 &#181;L in 180 &#181;L). The protocol followed the supplier&#8217;s recommendations:
	<ol>
		<li>Dilute samples and standard in diluent 2.</li>
		<li>Proceed to dilution of the standard in diluent 2 using a 1/4 serial fold dilution.</li>
		<li>Wash plates three times with wash buffer.</li>
		<li>Dispense 50 &#181;L of samples or standard per well.</li>
		<li>Incubate for 2 h at room temperature under agitation.</li>
		<li>Wash the plate four times with PBS + 0.05% Polysorbate 20.</li>
		<li>Add 25 &#181;L of detection antibody (60 &#181;L of each antibody for 3 mL final) in diluent 3.</li>
		<li>Incubate for 2 h at room temperature under agitation.</li>
		<li>Wash the plate four times with PBS + 0.05% Polysorbate 20.</li>
		<li>Avoiding bubbles, add 150 &#181;L per well of read buffer (Tris-based buffer containing tripropylamine, diluted 2x in ddH20) as a co-reactant for light generation in electrochemiluminescence immunoassays.</li>
		<li>Read the plate (without delay) on a multiplex plate reader.</li>
	</ol>
	</li>
	<li>Mouse TNF-a measurement by ELISA following the supplier&#8217;s protocol
	<ol>
		<li>Dilute supernatant 1:1 in assay diluent (ready to use protein-containing buffer).
		<ol>
			<li>Prepare reagents, samples, and standard dilutions as described in the kit. Add 50 &#956;L of assay diluent to each well.</li>
			<li>Add 50 &#956;L of standard, control, or sample per well.</li>
		</ol>
		</li>
		<li>Mix by gently tapping the plate frame for 1 min.</li>
		<li>Cover with the provided adhesive strip and incubate for 2 h at room temperature.</li>
		<li>Aspirate each well and wash with 400 &#956;L/well (repeat this step five times in total).</li>
		<li>After the final wash, remove any remaining wash buffer by aspirating.</li>
		<li>Invert the plate and blot against clean paper towels.</li>
		<li>Add 100 &#956;L of mouse TNF-&#945; conjugate to each well. Cover with a new adhesive strip.</li>
		<li>Incubate for 2 h at room temperature.</li>
		<li>Repeat the aspiration/wash as in step 7.4.4.</li>
		<li>Add 100 &#956;L of substrate solution to each well and incubate for 30 min at room temperature protected from light.</li>
		<li>Add 100 &#956;L of diluted hydrochloric acid solution (stop solution) to each well. Gently tap the plate to ensure thorough mixing.</li>
		<li>Measure the optical density at 450 nm (with the correction wavelength set at 560 nm) using a microplate reader (to be done within 30 min).</li>
	</ol>
	</li>
	<li>Cell viability
	<ol>
		<li>After removing supernatants from PBMC or monocyte preparations, assess cell viability using a ready to use resazurin solution (oxidation-reduction indicator) added directly to the cell suspension to 10% final concentration.</li>
		<li>Incubate for 1 to 2 h at 37 &#176;C, 5% CO2.</li>
		<li>Read fluorescence at 590 nm (Excitation 540 nm) using a microplate reader.</li>
	</ol>
	</li>
</ol>
8. Compound Preparation
<ol>
	<li>Serial dilution for iMoDCs
	<ol>
		<li>Dilute the MLT-827 stock solution (10 mM in dimethyl sulfoxide (DMSO) into medium to reach 8 &#181;M in one go (4x concentrated).</li>
		<li>Perform a six-step 1:5 serial dilution, using medium + 0.08% DMSO. Use the same medium + 0.08% DMSO solution for the vehicle (no compound) condition.</li>
	</ol>
	</li>
	<li>Single dose testing for iMoDCs
	<ol>
		<li>Dilute the MLT-827, AFN700 and Cpd11 stock solutions into medium to reach 4 &#181;M in one go (4x concentrated).</li>
	</ol>
	</li>
	<li>Serial dilution for murine spleen cells
	<ol>
		<li>Dilute a 10 &#181;M MLT-827 solution (obtained from a 10 mM stock solution following a one go dilution in medium) to 0.01 &#181;M, with a DMSO end concentration of 0.1%.</li>
		<li>For the dilution steps, take 2 &#181;L of each dilution, add 38 &#181;L RPMI, and pipet 5 &#181;L into the well. 
		NOTE: All treatments are performed in triplicate.</li>
	</ol>
	</li>
</ol>
9. Stimuli Preparation
<ol>
	<li>Depleted zymosan (DZ)
	<ol>
		<li>Add 2 mL of sterile endotoxin-free water to 10 mg of DZ.</li>
		<li>Vortex to homogenize the stock solution, vortex also prior each use.</li>
		<li>Aliquot solution and store aliquots at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Trehalose-6,6-dibehenate (TDB)
	<ol>
		<li>Add 100 &#181;L of DMSO to 1 mg TDB, heat at 60 &#176;C in a water bath for 15&#8211;30 s.</li>
		<li>Vortex and immediately add 900 &#181;L of sterile PBS, vortex again.</li>
		<li>Heat for 10&#8211;15 min at 60 &#176;C and homogenize by vortexing prior each use.</li>
		<li>Keep the solution at 4 &#176;C.</li>
		<li>Perform serial dilutions and single dose preparation as for the MLT-827 compound. 
		NOTE: Because TDB needs to be prepared in DMSO, a final 1% DMSO concentration is present during cell stimulation.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

In this work, we used simple experimental settings to study signaling pathways in human and mouse innate cells, and interrogate their dependency on MALT1 proteolytic function. Expanding on previous work11, our study showed that MALT1 paracaspase activity controls C-type lectin-like receptor induced cytokine production, including TNF-&#945;. In contrast, TLR-4-induced TNF-&#945; was independent of MALT1 in both species. Collectively, these data corroborated the key and selective contribution of the...

The paracaspase activity of MALT1 (Mucosa-associated lymphoid tissue lymphoma translocation protein 1) was revealed in 20081,2. Since then, a number of studies have reported its critical contribution to antigen receptor responses in lymphocytes. Genetic models in the mouse as well as pharmacology data support a key role in T cells, in T-cell dependent autoimmunity and in B-cell lymphoma settings3,4. In lymphocytes, MALT1 paracaspase activation occurs upon assembly of a CARD11-BCL10-MALT1 complex5, which is triggered by antigen-receptor proximal signaling downstream of the T- or B- cell receptor. There is also ample evidence that a similar CARD9-BCL10-MALT1 complex is important for propagating signals downstream of C-type lectin-like receptors (CLLR), e.g., Dectin-1, Dectin-2 and MINCLE in myeloid cells6,7. Dectin-1 has been particularly well studied because this pathway is critical for host defense against fungal infections8,9. Implication of MALT1 in Toll-like receptor (TLR) pathways, however, has remained controversial10. Recent evidence in human myeloid cells ruled out a direct role for MALT1 paracaspase activity in the regulation of TNF- production downstream of TLR-411.
In the present work, we used various experimental settings and stimulatory conditions in human and mouse myeloid cells to probe innate signaling pathways, relying on specific pharmacological tool inhibitors and measurement of cytokine production.

<table>
	<tbody>
		<tr>
			<td>100 &#181;m nylon cell strainer</td>
			<td>Sigma</td>
			<td>CLS431752</td>
			<td></td>
		</tr>
		<tr>
			<td>14 ml Falcon tube</td>
			<td>BD Falcon</td>
			<td>352057</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Falcon tube</td>
			<td>Falcon</td>
			<td>352090</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Falcon tube</td>
			<td>Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well plates</td>
			<td>Costar</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well flat-bottom plate, with low evaporation lid</td>
			<td>Costar</td>
			<td>3595</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well V-bottom plate</td>
			<td>Costar</td>
			<td>734-1798</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Chloride - NH4Cl</td>
			<td>Sigma</td>
			<td>A9434</td>
			<td></td>
		</tr>
		<tr>
			<td>Assay diluent RD1-W ELISA</td>
			<td>R&#38;D</td>
			<td>895038</td>
			<td>Assay diluent</td>
		</tr>
		<tr>
			<td>Cell culture microplate, 384 well, black</td>
			<td>Greiner</td>
			<td>781986</td>
			<td></td>
		</tr>
		<tr>
			<td>Depleted Zymosan</td>
			<td>Invivogen</td>
			<td>tlrl-dzn</td>
			<td>now: tlrl-zyd</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td>DMSO</td>
		</tr>
		<tr>
			<td>EDTA-Na2</td>
			<td>Sigma</td>
			<td>E5134</td>
			<td>Ethylenediaminetetraacetic acid disodium salt dihydrate</td>
		</tr>
		<tr>
			<td>ELISA muTNF-&#945;</td>
			<td>R&#38;D</td>
			<td>SMTA00</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll-Paque Plus</td>
			<td>GE Healthcare</td>
			<td>17-1440-03</td>
			<td></td>
		</tr>
		<tr>
			<td>gentleMACS C tubes</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-096-334</td>
			<td></td>
		</tr>
		<tr>
			<td>gentleMACS dissociator</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-093-235</td>
			<td></td>
		</tr>
		<tr>
			<td>GM-CSF</td>
			<td>Novartis</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat-inactivated Fetal bovine serum</td>
			<td>Gibco</td>
			<td>10082</td>
			<td>FBS</td>
		</tr>
		<tr>
			<td>HTRF hu IL-23</td>
			<td>CisBio</td>
			<td>62HIL23PEG</td>
			<td></td>
		</tr>
		<tr>
			<td>HTRF hu TNF-&#945;</td>
			<td>CisBio</td>
			<td>62TNFPEC</td>
			<td></td>
		</tr>
		<tr>
			<td>HTRF reconstitution buffer</td>
			<td>CisBio</td>
			<td>62RB3RDE</td>
			<td>50mM Phosphate buffer, pH 7.0, 0.8M KF, 0.2% BSA</td>
		</tr>
		<tr>
			<td>IFN-&#947;</td>
			<td>R&#38;D</td>
			<td>L4516</td>
			<td></td>
		</tr>
		<tr>
			<td>IL-4</td>
			<td>Novartis</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Abbott</td>
			<td>Forene</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipopolysaccharides (LPS)</td>
			<td>Sigma</td>
			<td>L4391</td>
			<td>LPS used in human samples</td>
		</tr>
		<tr>
			<td>Lipopolysaccharides</td>
			<td>Sigma</td>
			<td>L4516</td>
			<td>LPS used in murine samples</td>
		</tr>
		<tr>
			<td>Lysis buffer</td>
			<td>Self-made</td>
			<td>-</td>
			<td>155 mM NH4Cl, 10 mM KHCO3, 1 mM EDTA, pH 7.4</td>
		</tr>
		<tr>
			<td>Magnet</td>
			<td>Stemcell</td>
			<td>18001</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate, 384 well white</td>
			<td>Greiner</td>
			<td>784075</td>
			<td></td>
		</tr>
		<tr>
			<td>Monocytes enrichment kit</td>
			<td>Stemcell</td>
			<td>19059</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Mr. Frosty Cryo 1 &#176;C Freezing Container</td>
			<td>Nalgene</td>
			<td>5100-0001</td>
			<td>cooling device (containing Propanol-2)</td>
		</tr>
		<tr>
			<td>PBS 1x pH 7.4 [-] CaCl2 [-] MgCl2</td>
			<td>Gibco</td>
			<td>10010</td>
			<td>Phosphate-buffered saline</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>15140</td>
			<td>Pen/Strep</td>
		</tr>
		<tr>
			<td>Potassium bicarbonate - KHCO3</td>
			<td>Sigma</td>
			<td>P9144</td>
			<td></td>
		</tr>
		<tr>
			<td>PrestoBlue</td>
			<td>Invitrogen</td>
			<td>A13262</td>
			<td>Resazurin solution for viability assessment</td>
		</tr>
		<tr>
			<td>Propanol-2</td>
			<td>Merck</td>
			<td>1.09634</td>
			<td></td>
		</tr>
		<tr>
			<td>Read buffer</td>
			<td>MesoScale Discovery</td>
			<td>R92TC-3</td>
			<td>Tris-based buffer containing tripropylamine</td>
		</tr>
		<tr>
			<td>Recovery cell culture freezing medium</td>
			<td>Gibco</td>
			<td>12648-010</td>
			<td>freezing medium</td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute Medium (RPMI) with Glutamax</td>
			<td>Gibco</td>
			<td>61870</td>
			<td>+ 10% FBS for iMoDCs 
			+ 10% FBS + 1 mM Sodium Pyruvate + 100 U/mL Pen/Strep + 5 &#181;M &#946;-mercaptoethanol for human PBMCs and monocytes 
			+ 10% FBS + Pen/Strep + 5 &#181;M &#946;-mercaptoethanol for murine splenocytes</td>
		</tr>
		<tr>
			<td>Separation buffer</td>
			<td>Self-made</td>
			<td>-</td>
			<td>PBS pH 7.4 + 2% FBS + 1 mM EDTA pH 8.0</td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Gibco</td>
			<td>11360</td>
			<td></td>
		</tr>
		<tr>
			<td>Trehalose-6,6-dibehenate</td>
			<td>Invivogen</td>
			<td>tlrl-tdb</td>
			<td>TDB</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma</td>
			<td>P7949</td>
			<td>Polysorbate 20</td>
		</tr>
		<tr>
			<td>UltraPure 0.5 M EDTA pH 8.0</td>
			<td>Invitrogen</td>
			<td>15675</td>
			<td>Ethylenediaminetetraacetic acid</td>
		</tr>
		<tr>
			<td>Viewseal sealer</td>
			<td>Greiner BioOne</td>
			<td>676070</td>
			<td></td>
		</tr>
		<tr>
			<td>V-PLEX Proinflammatory Panel 1 Human Kit</td>
			<td>MesoScale Discovery</td>
			<td>K15049D</td>
			<td>electrochemiluminescent multiplex assay (IL-1&#946;, TNF-&#945;, IL-6, IL-8)</td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Gibco</td>
			<td>31350</td>
			<td></td>
		</tr>
	</tbody>
</table>

myeloid innate signaling pathway regulation by malt1 paracaspase activity

Besides its function in lymphoid cells, which has been addressed by numerous studies, the paracaspase MALT1 also plays an important role in innate cells downstream of pattern recognition receptors. Best studied are the Dectin-1 and Dectin-2 members of the C-type lectin-like receptor family that induce a SYK- and CARD9-dependent signaling cascade leading to NF-&#954;B activation, in a MALT1-dependent manner. By contrast, Toll-like receptors (TLR), such as TLR-4, propagate NF-&#954;B activation but signal via an MYD88/IRAK-dependent cascade. Nonetheless, whether MALT1 might contribute to TLR-4 signaling has remained unclear. Recent evidence with MLT-827, a potent and selective inhibitor of MALT1 paracaspase activity, indicates that TNF- production downstream of TLR-4 in human myeloid cells is independent of MALT1, as opposed to TNF- production downstream of Dectin-1, which is MALT1 dependent. Here, we addressed the selective involvement of MALT1 in pattern recognition sensing further, using a variety of human and mouse cellular preparations, and stimulation of Dectin-1, MINCLE or TLR-4 pathways. We also provided additional insights by exploring cytokines beyond TNF-, and by comparing MLT-827 to a SYK inhibitor (Cpd11) and to an IKK inhibitor (AFN700). Collectively, the data provided further evidence for the MALT1-dependency of C-type lectin-like receptor &#8212;signaling by contrast to TLR-signaling.

In myeloid cells, MALT1 relays activation signals downstream of several C-type lectin-like receptors, such as Dectin-1, Dectin-2 and MINCLE6. These pathways rely on (hem)ITAM motif-containing receptors (e.g., Dectin-1) or ITAM motif-containing co-receptors (e.g., FcR&#947;, for Dectin-2 and MINCLE) that recruit and activate the SYK kinase (Figure 1). This leads to activation of a protein kinase C isoform, namely PKC&#...

Watch this Scientific Journal Video about Myeloid Innate Signaling Pathway Regulation by MALT1 Paracaspase Activity at JoVE.com

Myeloid Innate Signaling Pathway Regulation by MALT1 Paracaspase Activity

MALT1 regulates innate immunity but how this occurs remains ill-defined. We used the selective MALT1 paracaspase inhibitor MLT-827 to unravel the contribution of MALT1 to innate signaling downstream of Toll-like or C-type lectin-like receptors, demonstrating that MALT1 regulates the production of myeloid cytokines, and downstream of C-type lectin-like receptors, selectively.

Besides its function in lymphoid cells, which has been addressed by numerous studies, the paracaspase MALT1 also plays an important role in innate cells ...

myeloid-innate-signaling-pathway-regulation-malt1-paracaspase

Research

JoVE Journal

Biology

7.4K Views.  Novartis Institutes for BioMedical Research. MALT1 regulates innate immunity but how this occurs remains ill-defined. We used the selective MALT1 paracaspase inhibitor MLT-827 to unravel the contribution of MALT1 to innate signaling downstream of Toll-like or C-type lectin-like receptors, demonstrating that MALT1 regulates the production of myeloid cytokines, and downstream of C-type lectin-like receptors, selectively.

Video: Myeloid Innate Signaling Pathway Regulation by MALT1 Paracaspase Activity

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

Preparation of 2-dGuo-Treated Thymus Organ Cultures

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play a key role in the development of a functionally competent T-cell pool. However, the complexity of T-cell development in the thymus in vivo can limit analysis of individual cellular components and particular stages of development. In vitro culture systems provide a readily accessible means to study multiple complex cellular processes. Thymus organ culture systems represent a widely used approach to study intrathymic development of T-cells under defined conditions in vitro. Here we describe a system in which mouse embryonic thymus lobes can be depleted of endogenous haemopoeitic elements by prior organ culture in 2-deoxyguanosine, a compound that is selectively toxic to haemopoeitic cells. As well as providing a readily accessible source of thymic stromal cells to investigate the role of thymic microenvironments in the development and selection of T-cells, this technique also underpins further experimental approaches that include the reconstitution of alymphoid thymus lobes in vitro with defined haemopoietic elements, the transplantation of alymphoid thymuses into recipient mice, and the formation of reaggregate thymus organ cultures. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).

This video demonstrates the dissection and removal of the fetal thymus as well the preparation of ex vivo cultures of 2-dGuo-treated thymus.

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play ...

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative stress refers to the aberrant formation of ROS (reactive oxygen species), which include O2&bull;-, H2O2, and hydroxyl radicals. Reactive oxygen species influences a multitude of cellular processes including signal transduction, cell proliferation and cell death1-6. ROS have the potential to damage vascular and organ cells directly, and can initiate secondary chemical reactions and genetic alterations that ultimately result in an amplification of the initial ROS-mediated tissue damage. A key component of the amplification cascade that exacerbates irreversible tissue damage is the recruitment and activation of circulating inflammatory cells. During inflammation, inflammatory cells produce cytokines such as tumor necrosis factor-&alpha; (TNF&alpha;) and IL-1 that activate endothelial cells (EC) and epithelial cells and further augment the inflammatory response7. Vascular endothelial dysfunction is an established feature of acute inflammation. Macrophages contribute to endothelial dysfunction during inflammation by mechanisms that remain unclear. Activation of macrophages results in the extracellular release of O2&bull;- and various pro-inflammatory cytokines, which triggers pathologic signaling in adjacent cells8. NADPH oxidases are the major and primary source of ROS in most of the cell types. Recently, it is shown by us and others9,10 that ROS produced by NADPH oxidases induce the mitochondrial ROS production during many pathophysiological conditions. Hence measuring the mitochondrial ROS production is equally important in addition to measuring cytosolic ROS. Macrophages produce ROS by the flavoprotein enzyme NADPH oxidase which plays a primary role in inflammation. Once activated, phagocytic NADPH oxidase produces copious amounts of O2&bull;- that are important in the host defense mechanism11,12. Although paracrine-derived O2&bull;- plays an important role in the pathogenesis of vascular diseases, visualization of paracrine ROS-induced intracellular signaling including Ca2+ mobilization is still hypothesis. We have developed a model in which activated macrophages are used as a source of O2&bull;- to transduce a signal to adjacent endothelial cells. Using this model we demonstrate that macrophage-derived O2&bull;- lead to calcium signaling in adjacent endothelial cells.

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

An efficient method to gain insights into visualizing the paracrine-derived ROS induction of endothelial Ca2+ signaling is described. This method takes advantage of measuring paracrine derived ROS triggered Ca2+ mobilization in vascular endothelial cells in a co-culture model.

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning

Signal transduction by growth factor receptors is essential for cells to maintain proliferation and differentiation and requires tight control. Signal transduction is initiated by binding of an external ligand to a transmembrane receptor and activation of downstream signaling cascades. A key regulator of mitogenic signaling is Grb2, a modular protein composed of an internal SH2 (Src Homology 2) domain flanked by two SH3 domains that lacks enzymatic activity. Grb2 is constitutively associated with the GTPase Son-Of-Sevenless (SOS) via its N-terminal SH3 domain. The SH2 domain of Grb2 binds to growth factor receptors at phosphorylated tyrosine residues thus coupling receptor activation to the SOS-Ras-MAP kinase signaling cascade. In addition, other roles for Grb2 as a positive or negative regulator of signaling and receptor endocytosis have been described. The modular composition of Grb2 suggests that it can dock to a variety of receptors and transduce signals along a multitude of different pathways1-3.
	Described here is a simple microscopy assay that monitors recruitment of Grb2 to the plasma membrane. It is adapted from an assay that measures changes in sub-cellular localization of green-fluorescent protein (GFP)-tagged Grb2 in response to a stimulus4-6. Plasma membrane receptors that bind Grb2 such as activated Epidermal Growth Factor Receptor (EGFR) recruit GFP-Grb2 to the plasma membrane upon cDNA expression and subsequently relocate to endosomal compartments in the cell. In order to identify in vivo protein complexes of Grb2, this technique can be used to perform a genome-wide high-content screen based on changes in Grb2 sub-cellular localization. The preparation of cDNA expression clones, transfection and image acquisition are described in detail below. Compared to other genomic methods used to identify protein interaction partners, such as yeast-two-hybrid, this technique allows the visualization of protein complexes in mammalian cells at the sub-cellular site of interaction by a simple microscopy-based assay. Hence both qualitative features, such as patterns of localization can be assessed, as well as the quantitative strength of the interaction.

A high-content screening method for the identification of novel signaling competent transmembrane receptors is described. This method is amenable to large-scale automation and allows predictions about in vivo protein binding and the sub-cellular localization of protein complexes in mammalian cells.

Signal  transduction by growth factor receptors is essential for cells to maintain  proliferation and differentiation and requires tight control. Signal  ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...